Experimental evidence of phospho enolpyruvate resynthesis ...13 13C-3-pyruvate feeding suggests that...
Transcript of Experimental evidence of phospho enolpyruvate resynthesis ...13 13C-3-pyruvate feeding suggests that...
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Experimental evidence of phosphoenolpyruvate resynthesis from pyruvate in illuminated leaves Guillaume Tcherkez1,2,3*, Aline Mahé1, Edouard Boex-Fontvieille1, Elisabeth Gout4, Florence Guérard2 and Richard Bligny4 1. Institut de Biologie des Plantes, CNRS UMR 8618, Université Paris-Sud 11, 91405 Orsay cedex, France. 2. Plateforme Métabolisme-Métabolome, IFR 87, Batiment 630, Université Paris-Sud 11, 91405 Orsay cedex. 3. Institut Universitaire de France, 103 boulevard Saint-Michel, 75005 Paris, France. 4. Laboratoire de Physiologie Cellulaire Végétale, UMR 5168, CEA Grenoble, 38054 Grenoble cedex 9, France. *Corresponding author: [email protected]; fax. +33 1 69 15 34 24. Key words: day respiration, labeling, NMR, phosphoenolpyruvate, glutamate. Number of words: 4,999 (incl. abstract, without references and figure legends).
Plant Physiology Preview. Published on July 5, 2011, as DOI:10.1104/pp.111.180711
Copyright 2011 by the American Society of Plant Biologists
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Abstract (190 words) 1 2 Day respiration is the cornerstone of N assimilation since it provides carbon skeletons to 3
primary metabolism for glutamate and glutamine synthesis. However, recent studies have 4
suggested that the tricarboxylic acid pathway (TCAP) is rate-limiting and mitochondrial 5
pyruvate dehydrogenation is partly inhibited in the light. Pyruvate may serve as a carbon 6
source for amino acid (e.g. alanine) or fatty acid synthesis but pyruvate metabolism is not 7
well documented and neither is the possible resynthesis of phosphoenolpyruvate (PEP). Here, 8
we examined the capacity of pyruvate to convert back to PEP using 13C and 2H labeling in 9
illuminated cocklebur (Xanthium strumarium) leaves. We show that the intramolecular 10
labeling pattern in glutamate, 2-oxoglutarate and malate after 13C-3-pyruvate feeding was 11
consistent with 13C-redistribution from PEP via the PEP-carboxylase reaction. Furthermore, 12
the deuterium loss in glutamate after 2H3-13C-3-pyruvate feeding suggests that conversion to 13
PEP and back to pyruvate washed out 2H atoms to the solvent. Our results demonstrate that in 14
cocklebur leaves, PEP resynthesis occurred as a flux from pyruvate, ca. 0.5‰ of the net CO2 15
assimilation rate. This is likely to involve pyruvate Pi dikinase and the fundamental 16
importance of this flux for PEP and Pi homeostasis is discussed. 17
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Introduction 19
It is now 63 years that Kok decribed the effect by which non-photorespiratory CO2 evolution 20
increases at low light levels, providing evidence for the inhibition of leaf respiration by light 21
(Kok 1948). Since then, respiration of illuminated leaves (‘day respiration’) and its inhibition 22
has been extensively characterized with gas-exchange and metabolic analyses (for a review, 23
see Atkin et al. 2000). Recent papers have further emphasised the complex metabolic 24
relationships between day respiration and other metabolic processes such as nitrogen 25
metabolism, since carbon skeletons synthesised by the tricarboxylic acid pathway (TCAP) are 26
essential for N assimilation (Paul and Pellny 2003, Nunes-Nesi et al. 2007). The metabolic 27
mechanisms by which respiratory metabolism is down-regulated in the light has been 28
summarized previously (Tcherkez et al. 2009). Essentially, the activity of the TCAP has been 29
shown to be strongly inhibited (Hanning and Heldt 1993, Tcherkez et al. 2005, Gauthier et al. 30
2010) and therefore, the consumption of pyruvate molecules synthesised in the light by the 31
TCAP is believed to be limited. 32
This raises the question of the fate of pyruvate and acetyl-CoA produced by the 33
pyruvate dehydrogenase (PDH; Figure 1, arrow 1). Acetyl-CoA is not likely to accumulate. 34
Firstly, the PDH is retro-inhibited by its product acetyl-CoA (Harding et al. 1970, Rapp et al. 35
1987) and PDH activity is down-regulated by phosphorylation (Budde and Randall 1990, 36
Tovar-Mendez et al. 2003). This effect may in turn contribute to the build-up of the pyruvate 37
pool. Secondly, a significant fraction of acetyl-CoA is directed to fatty acids production in the 38
chloroplast (Ohlrogge and Jaworski 1997). Accordingly, the antisense line of Arabidopsis 39
associated with PDH kinase (in which the mitochondrial PDH reaction is thus enhanced) 40
accumulated 14C-labeled fatty acids when 14C-Pyr was fed to photosynthetic stems (Marillia et 41
al. 2003). 42
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By contrast, pyruvate is believed to accumulate to some extent in the light. Metabolic 43
measurements in leaves along a day/night cycle have shown that the pyruvate content is 44
roughly two-fold larger in the light (Scheible et al. 2000). Pyruvate is also converted to 45
alanine, as shown by 13C-labeling (Tcherkez et al. 2005; Figure 1, arrow 2). That said, 46
pyruvate production itself (by pyruvate kinase, EC 2.7.1.40; Figure 1, arrow 3) is believed to 47
be partly inhibited in the light, due to the regulatory properties of the enzyme. In tobacco 48
(Nicotiana tabacum) leaves, the total activity of pyruvate kinase has been shown to be lower 49
in the light compared to the dark (Scheible et al. 2000) and in spinach (Spinacia oleracea) 50
chloroplasts, the pyruvate content appeared lower in the light than in the dark (Santarius and 51
Heber 1965). In the alga Selenastrum minutum, the chloroplastic enzyme is inhibited by 52
photosynthetic intermediates (e.g. ribulose-1,5-bisphosphate) and the cytosolic enzyme is 53
inhibited by Pi and glutamate (Lin et al. 1989). Furthermore, leaf enzymes are inhibited by 54
citrate or aspartate (Baysdorfer and Bassham 1984) and activated by dihydroxyacetone 55
phosphate (Lin et al. 1989). Therefore in the light, pyruvate kinase activity is presumed to be 56
strongly down-regulated in chloroplasts and finely adjusted (by the balance between upstream 57
and downstream metabolites) in the cytosol. 58
Still, the reverse reaction, that is, resynthesis of phosphoenolpyruvate (PEP) from 59
pyruvate (Figure 1, arrow 4), is not well documented. In principle, the pyruvate kinase is 60
reversible (Krimsky 1959, Robinson and Rose 1972, Dyson et al. 1975), but under ordinary 61
conditions, the reaction has a large equilibrium constant towards pyruvate production 62
(McQuate and Utter 1959, Krimsky 1959, Nageswara-Rao et al. 1979 and see Table S1). The 63
reverse reaction, if it happens to occur, may be assumed to rather involve the pyruvate Pi 64
dikinase (EC 2.7.9.1). This enzyme has recently been found and characterised in Arabidopsis 65
leaves, in which there are two isoforms (cytosolic and chloroplastic) (for a review, see 66
Chastain and Chollet 2003). Furthermore, a light/dark regulation of the leaf enzyme involving 67
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phosphorylation has been demonstrated (Chastain et al. 2002). However, the function of the 68
enzyme remains enigmatic. Some data suggest a role in the response to stress such as anoxia 69
(Lasanthi-Kuddahettige et al. 2007, Doubnerova and Ryslava 2011). In seeds, PPDK activity 70
seems essential in controlling starch biosynthesis by providing cytosolic PPi and hexose 71
phosphates via reversed glycolysis (Kang et al. 2005). In leaves, substantial mid-vein PPDK 72
activity has been demonstrated, suggesting a role in providing PEP to the shikimate pathway 73
for lignin biosynthesis (Hibberd and Quick 2002). Transcripts encoding cytosolic PPDK have 74
been shown to increase during senescence of Arabidopsis leaves (Lin and Wu 2004, Taylor et 75
al. 2010) so that PPDK activity has been assumed to participate to nitrogen remobilization 76
associated with leaf senescence (Taylor et al. 2010): PEP synthesized from pyruvate by PPDK 77
is believed to be converted to organic acids via PEP-carboxylase thus sustaining the 78
production of the transport amino acid glutamine. 79
Here, we examined the metabolic flux associated with the possible conversion of 80
pyruvate back to PEP in illuminated leaves, using strong isotopic labeling with protio-13C- 81
and deutero-13C-pyruvate (2H3-13C-3-pyruvate, thereafter denoted as d-13C-3-pyruvate). The 82
fate of 13C-atoms was then followed by nuclear magnetic resonance (NMR). We took 83
advantage of the contrasted enrichment pattern expected when conversion back to PEP is 84
involved (Figure 1): PEP resynthesis followed by PEP carboxylation mostly redistributes the 85
13C label in the C-2 atom position of glutamate while the 13C-3-pyruvate consumption by the 86
TCAP produces 13C-4-glutamate. We show that pyruvate can be converted back to PEP 87
although the associated flux is quantitatively minor. Our data thus suggest that PEP 88
resynthesis contributes to consuming pyruvate molecules that tend to accumulate in the light. 89
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Results 91
13C-labeling pattern in metabolites 92
The 13C-labeling pattern in major metabolites after 13CO2 and/or 13C-3-pyruvate feeding is 93
shown in Figure 2. As expected, 13C-pyruvate labeling caused an isotopic enrichment 94
(positional % 13C) up to 15% in glutamate C-4 and glutamine C-4, and 6% in isocitrate C-4, 95
reflecting the consumption of the 13C-label by the TCAP. There is also a clear 13C-enrichment 96
in malate C-3 (15%) and glutamate C-3 (3.5%) (and also glutamate C-2, see the next section), 97
suggesting either the involvement of the production of malate from PEP resynthesised from 98
13C-3-pyruvate, or several rounds of the TCAP cycle (Figure 1). However, succinate and 99
fumarate were not labeled, strongly suggesting that most 13C-atoms were not committed into 100
the full cycle, that is, multiple rounds cannot account for the neat labeling in malate C-3. 13C-101
3-pyruvate was also consumed by amino acid synthesis, such as alanine (3% 13C in C-3) and 102
valine (34% 13C in methyl groups). The labeling was accompanied by a small refixation of 103
13CO2 produced by the decarboxylation of 13C-3-pyruvate (by the TCA cycle). Such a 104
refixation was very small, with 5% 13C in furano-fructose C-2 and 3% 13C in both α-glucose 105
and the glucosyl moiety of sucrose. Using metabolite contents, this demonstrates that 13C-106
refixation was, at most, of 6% of the TCAP 13C-commitment to glutamate. 107
When leaves were labeled with 13CO2, most sugars showed a clear 13C-enrichment, as 108
well as malate and aspartate C-2, showing the involvement of the PEP carboxylase (PEPc) 109
reaction from 13C-2-enriched PEP that was in turn formed by glycolysis. When both 13C-3-110
pyruvate and 13CO2 were used, the effect of the double labeling was not simply additive. In 111
fact, the C-1 in malate was clearly labeled (9% 13C), suggesting 13C-bicarbonate fixation by 112
the PEPc in C-4 and subsequent equilibration to C-1 via fumarate; similarly, C-1, C-2 and C-3 113
positions in glutamate were labeled (up to 11%), indicating the consumption of malate formed 114
from 13C-enriched PEP. 115
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Specific 13C-pattern in glutamate 116
The specific 13C-enrichment pattern in glutamate is detailed in Figure 3. The positional 13C-117
abundance found in glutamate C-1 upon 13C-3-pyruvate labeling was 1.7%, very close to the 118
natural abundance (1.1%, dashed line in Figure 3B). The 13C-abundance in C-2 and C-3 was 119
3-4%, showing the redistribution of the 13C-label. The % 13C in C-4 was very high because it 120
inherited the 13C-label via the TCAP (Figure 1). With 13CO2, the reverse occurred, the C-4 121
position being at natural abundance while the C-1 to C-3 positions were labeled 3-4% 13C. 122
The positional labeling in glutamate was associated with {13C-13C} interactions 123
between neighbour carbon atoms (Figure 3C). Upon 13C-3-pyruvate labeling, the most labeled 124
position (C-4) was associated with little interaction (6% only of 13C-4 atoms were 125
accompanied by a 13C-neighbour) while the interaction was 62% in C-3. In other words, most 126
13C-3-glutamate molecules were also labeled on a neighbour carbon while most 13C-4-127
glutamate molecules were not labeled on a neighbour carbon. This indicates that presumably, 128
the consumption of 13C-2-oxaloacetate molecules by the TCAP mostly used 13C-2-acetyl-CoA 129
while the reciprocal was not true (most 13C-2-acetyl-CoA molecules did not react with 13C-2-130
oxaloacetate). The C-2 atom position in glutamate did not show visible {13C-13C} interaction. 131
Upon 13CO2 labeling, there were substantial {13C-13C} interactions, with the largest 132
value in C-2, suggesting that 13C-2-oxaloacetate molecules were clearly coupled to the input 133
of 13C-2-acetyl-CoA. Similarly, double labeling with 13CO2 and 13C-3-pyruvate lead to nearly 134
100% {13C-13C} interaction in C-3 with a clear double interaction (on both C-2 and C-4 135
sides), showing the concerted contribution of 13C-2-oxaloacetate and 13C-2-acetyl-CoA to 136
produce glutamate. Furthermore, the largest interaction values were obtained with C-2 and C-137
3 positions (77 and 94% vs 46% in C-4), suggesting the involvement of 13C2-2,3-oxaloacetate 138
(Figure 1). In 2-oxoglutarate, significant {13C-13C} interaction was only visible in C-4 (see 139
also Supplementary material, Figure S1), with a larger value upon double labeling, that was 140
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consistent with that found in glutamate C-4. The C-2 position in 2-oxoglutarate corresponds 141
to the C=O (keto) C-atom and was therefore difficult to analyse by NMR because of long 142
relaxation time, about 30 s (remote chemical shift region), that caused an under-estimation of 143
corresponding resonance peaks. That said, there appeared to be no substantial labeling in that 144
position (Supplementary material, Figure S1). 145
146
Proton exchange in glutamate C-4 147
13C-labeling was carried out with either protonated (CH3-CO-COO–) or deuterated (50% CD3-148
CO-COO–) pyruvate. The labeling pattern in metabolites upon d-13C-3-pyruvate was very 149
similar to that in Figure 2 (not shown). The resolution and intensity of deuterated 13C-150
pyruvate were not large enough to appreciate deuterium content directly on the pyruvate pool. 151
Therefore, the fate of deuterium was followed using the {13C-D} interaction on glutamate C-152
4. The NMR signal obtained is shown in Figure 4. Clearly, a significant fraction of 13C-4-153
glutamate molecules were also deuterated, causing a change of around 0.25 ppm (28 Hz) on 154
the 13C chemical shift, with the appearance of a triplet. Deuterated glutamate was found to 155
represent 33% of total 13C-4-glutamate. That value is significantly less than that in source 156
pyruvate (50%), suggesting a loss of deuterium during metabolism of pyruvate to glutamate. 157
This effect may have come from either the H/D isotope effect of citrate synthase (the other 158
two first enzymes of the TCA cycle, aconitase and isocitrate dehydrogenase, do not involve 159
the CH2-protons along their mechanism) or the wash-out of deuterium atoms to the solvent 160
due to pyruvate enolisation. However, we found no significant isotope effect on the 13C-161
amount in glutamate (37 and 41 µmol m-2 [internal standard equivalents] with deutero- and 162
protio-13C-3-pyruvate, respectively), showing that the rate of 13C-3-pyruvate incorporation by 163
citrate synthase was not slowed down by deuterium under our conditions. The loss of 164
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deuterium was thus caused by isotopic wash-out associated with pyruvate conversion to 165
phosphoenolpyruvate. 166
167
Pyruvate Pi dikinase activity and phosphometabolites 168
Total leaf pyruvate Pi dikinase activity was measured from illuminated leaves and also during 169
the night (1 h 30 dark) (Figure 5C). The activity was clearly lower in the dark than in the 170
light. Still, the in vitro activity value found here (0.04 µmol m-2 s-1) in the light was very small 171
as compared to photosynthesis (near 10 µmol m-2 s-1, not shown). There was a significant 172
decrease (two-fold) of the pyruvate content in the dark while the PEP content decreased a bit 173
less, from 2.9 to 1.7 µmol m-2 (Figure 5A). Leaf ATP/ADP ratio decreased from around 2.5 in 174
the light to 1.2 in the dark and the ATP/AMP ratio remained unchanged (around 3) (Figure 175
5B). Therefore, the mass action ratio of pyruvate kinase ([pyruvate/PEP]×[ATP/ADP]) 176
appeared to be less favourable to the reaction in the light than in the dark. The corresponding 177
mass action ratio of pyruvate Pi dikinase ([PEP/pyruvate]×[AMP/ATP]) was less favourable 178
to the reaction in the dark, provided pyrophosphate (PPi) remained constant (not measured 179
here, not visible on NMR analyses). 180
The natural co-variation in phosphometabolites was investigated, using 6 replicates of 181
leaves sampled in identical photosynthetic conditions, with pyruvate feeding (Figure 6). 182
Hierarchical clustering analysis shows that phosphoenolpyruvate was in the same group as 183
AMP, ADP, fructose-1,6-bisphosphate, ribulose-1,5-bisphosphate, ATP and ADP-glucose 184
(group II, Figure 6C), that is, primary photosynthetic phosphometabolites. That is, net PEP 185
production co-varied tightly with chloroplastic photosynthetic activity (rather than cytosolic 186
metabolites such as UDP-glucose). PEP/AMP and PEP/fructose-1,6-bisphosphate ratios were 187
rather constant (Figure 6B), for any UDP-glucose/UTP values. Furthermore, when plotted 188
against fructose-1,6-bisphosphate, [PEP×AMP]/[ATP] increased and so did the 189
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[PEP×ADP]/[ATP] ratio. In other words, there was a positive relationship between the mass 190
action ratio of pyruvate kinase and the phospho-hexose content. 191
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Discussion 193
The metabolism of pyruvate in illuminated C3 leaves is not very well documented and much 194
uncertainty remains on metabolic fluxes associated with the different pathways that consume 195
pyruvate. Here, we used isotopic labeling to investigate whether the resynthesis of PEP from 196
pyruvate occurred in the light, by the involvement of, e.g., pyruvate Pi dikinase. First, we 197
carried out labeling with 13C-3-pyruvate to trace the possible redistribution of 13C in C-atom 198
positions derived from PEP C-3 (Figure 1). Second, we used deuterated 13C-3-pyruvate 199
(double labeling) to examine whether deuterium atoms were preserved in 13C-4-glutamate. 200
Third, we measured leaf pyruvate Pi dikinase activity and phosphometabolites so as to better 201
understand PEP metabolism. 202
203
Is pyruvate converted back to PEP? 204
After 13C-3-pyruvate labeling, we found a clear labeling in C-atom positions (glutamate C-4, 205
glutamine C-4, isocitrate C-4) inherited from the TCAP activity that consumes 13C-2-acetyl-206
CoA formed from pyruvate. Such results are consistent with those obtained in cocklebur 207
(Xanthium strumarium) in similar conditions (Tcherkez et al. 2008, 2009) and show that 208
glutamate was one major sink metabolite after pyruvate addition (nearly 30% of total non-209
sugar 13C in samples). Importantly, 13C atoms were also redistributed in positions inherited 210
from PEP, such as malate C-3. Furthermore, while not very visible, oxaloacetate showed a 211
very slight increase from 1.4 to 2% 13C in C-3 (Figure 2). 212
It remains possible that 13C-3-pyruvate caused a labeling in malate and oxaloacetate 213
via several rounds of the TCAP (Krebs ‘cycle’). However, under such an assumption, the 214
isotopic pattern in malate would show a larger enrichment in C-2 than in C-3. Here, malate C-215
2 is not labeled (Figure 2). In addition, previous studies in cocklebur showed the 216
accumulation of fumarate and glutamate, with no possible 13C-labeling in succinate, thereby 217
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showing the non-cyclic nature of the TCAP (Tcherkez et al. 2009) with a 5-10% recovery of 218
2-oxoglutarate back to citrate through the cycle. Furthermore, the isotopic pattern found in 219
glutamate was not strictly consistent with a cycle-based 13C-redistribution. We show that the 220
probability to observe a neighbour 13C associated with glutamate C-4 is 6-10% (Figure 3). 221
Assuming this value represents the 13C-commitment to TCAP cyclicity, the isotopic pattern 222
expected in glutamate would be C-4, 15% (observed value), C-3, 2.6% (= [15–1.1]×0.1+1.1) 223
and C-2, 1.2% (= [2.6–1.1]×0.1+1.1). We obtained here larger 13C-enrichment values (4.5 and 224
2.5% in C-3 and C-2, respectively), suggesting other pathways fed the TCAP with 13C. We 225
therefore believe that the contribution of TCAP cyclicity to 13C-patterns found here was of 226
little importance under our conditions. 227
The involvement of the conversion of pyruvate back to PEP further agrees with the 228
{13C-13C} interactions between atom positions in glutamate. Although slight (near 4% 13C), 229
labeling in glutamate C-3 was associated with that in C-4 and C-2 (62% interaction), showing 230
the concerted input of 13C-oxaloacetate (formed from 13C-PEP) and 13C-acetyl-CoA upon 13C-231
3-pyruvate feeding (Figures 1 and 3). 232
When fed with d-13C-3-pyruvate, the deuterium content in glutamate C-4 is lower 233
(34%) than that in source pyruvate (50%). Since there was no H/D isotope effect associated 234
with the 13C-commitment rate under our conditions, such a loss of deuterium likely came 235
from the isotopic wash-out to the solvent caused by the keto-enol equilibrium of pyruvate. 236
The spontaneous equilibrium in water disfavours strongly the enol form (Keq 2⋅10-5, Esposito 237
et al. 1999), with a very low rate constant of keto-to-enol conversion (around 10-6 s-1). Such a 238
value would give, in our 3-h experiment, an estimated proton exchange with the solvent of 10-239
6 s-1×15⋅10-3 mol L-1×3×3600 = 0.16 mmol L-1 experiment-1 that is, 1% only of source 240
pyruvate concentration. In other words, most of the deuterium loss in glutamate C-4 was due 241
to metabolic (enzymatic) effects, demonstrating that 13C-3-pyruvate molecules underwent 242
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enolisation, likely back to phosphoenolpyruvate. This process was nevertheless a dynamic 243
equilibrium that reformed pyruvate so that glutamate C-4 was only partially deuterated. 244
The metabolic flux associated with the conversion of pyruvate back to PEP may be 245
roughly estimated with the 13C-enrichment pattern in glutamate C-4 and C-2 (Figure 1). The 246
13C-percentage excess (compared to natural abundance) in C-4 was nearly 15 – 1.1 ~ 14% and 247
that in C-2 was 3 – 1.1 ~ 2%. Neglecting multiple rounds of the TCAP cycle, the relative 248
commitment of pyruvate to PEP and oxaloacetate was then 2/14 = 14% of the commitment of 249
pyruvate to the TCAP via acetyl-CoA. Tcherkez et al. (2008) estimated that under similar 250
conditions (pyruvate feeding in the light), the pyruvate-decarboxylating TCAP activity is 0.02 251
µmol m-2 s-1 and so the flux associated with conversion back to PEP would be near 0.002 252
µmol m-2 s-1. In intact illuminated leaves, assuming a day respiration rate of 0.5 µmol m-2 s-1 253
and a natural TCAP activity of around 0.05 µmol m-2 s-1 (near 10% of the day CO2 evolution 254
rate), PEP resynthesis would be of 0.005 µmol m-2 s-1 (say, roughly 0.5‰ only of the net 255
assimilation rate). 256
257
Which is the enzyme of PEP/pyruvate metabolism? 258
PEP resynthesis from pyruvate may involve pyruvate kinase or pyruvate Pi dikinase. Pyruvate 259
kinase has been shown to be reversible, with a huge equilibrium constant of 1011.5 towards 260
pyruvate production (nearly irreversible) (Nageswara-Rao et al. 1979 and Table S1). Still, 261
proton exchange between pyruvate and solvent water has been demonstrated (Robinson and 262
Rose 1972, Rose et al. 1991), showing the ability of the enzyme to catalyse the reverse 263
reaction. However, the mass action ratio [pyruvate×ADP]/[PEP×ATP] is less favourable in 264
the light than in the dark, with a larger leaf ATP/ADP ratio in the light (Figure 5). Subcellular 265
analysis of barley protoplasts (Hordeum vulgare) also showed that the ATP/ADP ratio was 266
larger in both chloroplast and cytosol in the light (Gardeström 1987, but see Stitt et al. 1982), 267
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thereby disfavouring the pyruvate kinase activity, which is inhibited by ATP (Baysdorfer and 268
Bassham 1984). In vitro studies have further shown that major compounds such as glutamate 269
inhibit cytosolic pyruvate kinase and photosynthetic intermediates inhibit chloroplastic 270
pyruvate kinase (Lin et al. 1989). It is therefore believed that both chloroplastic and cytosolic 271
pyruvate kinase activity is down-regulated in illuminated leaves (Plaxton 1996). 272
By contrast, chloroplastic pyruvate Pi dikinase (PPDK) is activated in the light due to 273
its reversible dephosphorylation by Pi, which is in turn converted to pyrophosphate (PPi) 274
(Chastain et al. 2002, Chastain and Chollet 2003). The inactivation of the enzyme involves 275
phosphorylation by ADP, converted to AMP. Therefore, both Pi/PPi and ADP/AMP ratios 276
may be of importance to control PPDK (de)phosphorylation. Furthermore, such ratios have a 277
thermodynamic effect on the reaction catalyzed by PPDK, the Keq of which is around 100 278
only (Table S1). That is, the reaction is much more reversible than pyruvate kinase. We found 279
a ADP/AMP ratio of 1.3 and 2.3 in the light and in the dark, respectively (Figure 5). In 280
illuminated isolated chloroplasts, a low ADP/AMP ratio was observed while the Pi 281
concentration was quite high (up to 25 mmol L-1) (Santarius and Heber 1965) and PPi 282
concentration was believed to be very low (Edwards et al. 1978). It is thus likely that both the 283
dephosphorylation of PPDK and the PEP-forming reaction of the equilibrium are promoted by 284
light in chloroplasts. In the cytosol, Pi concentration is certainly lower, less than 1 millimolar 285
(Bligny et al. 1990) and more probably near 70 µmol L-1 (Pratt et al. 2009). By contrast, 286
pyrophosphate is predominantly in the cytosol, up to 0.3 mmol L-1 (Weiner et al. 1987), 287
possibly due to the absence of cytosolic inorganic pyrophosphatase (Farré et al. 2006, but see 288
Rojas-Beltran et al. 1999). Furthermore, the cytosolic ATP/AMP ratio is similar (7-8) in the 289
light and in the dark (Stitt et al. 1982). The PPDK-catalysed reaction is thus slightly less 290
favourable in the cytosol than in the chloroplast under illumination, but may still be driven by 291
the very large ATP/AMP ratio. 292
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We thus hypothesise here that the conversion of pyruvate back to PEP involves PPDK 293
rather than pyruvate kinase. Accordingly, our results indicate that the leaf PPDK activity in 294
the light is larger than in the dark (Figure 5), as already shown elsewhere (Chastain and 295
Chollet 2003). 296
297
Rationale of PEP resynthesis from pyruvate 298
Leaf PPDK activity is influenced by light/dark conditions (see just above) and therefore, the 299
biological significance of the PPDK-flux is certainly related to the control of day respiration 300
and pyruvate metabolism. We nevertheless recognize that at the leaf level, PPDK activity is 301
believed to be heterogenous, with larger values in vein tissues (Hibberd and Quick 2002). It is 302
therefore possible that the PPDK activity observed here was partly associated with vascular 303
cells, which require PEP for lignin biosynthesis. However, the labeling in major metabolites 304
associated with PEPc-catalyzed carbon fixation (malate) or nitrogen fixation (glutamate) was 305
very clear (Figure 2), suggesting a wider implication of the PPDK-flux in cocklebur leaves. In 306
fact, PEP resynthesis by the PPDK has been assumed to be involved in nitrogen assimilation. 307
First, in senescing Arabidopsis leaves, the conversion of pyruvate back to PEP sustains 308
oxaloacetate synthesis via the PEPc, thereby promoting glutamine production for nitrogen 309
remobilization (Taylor et al. 2010). Second, transcripts encoding PPDK have been reported to 310
be more abundant in roots of rice (Oryza sativa) lines over-expressing alanine 311
aminotransferase (AlaAT) as compared to the wild-type (Beatty et al. 2009). Since AlaAT 312
promotes nitrogen uptake and subsequent metabolism by the downstream catalysis of alanine 313
to pyruvate (with the concomitant usage of 2-oxoglutarate and the formation of Glu), this 314
suggests a role of PPDK in the recovery of oxaloacetate for glutamate production. 315
In illuminated leaves, the PPDK activity may have further roles. As stated in the 316
Introduction, cytoplasmic pyruvate consumption is believed to be lower in the light compared 317
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16
to the dark, due to the partial inhibition of the PDH and the substantial inhibition of the 318
TCAP. It is therefore plausible that PEP resynthesis contributes to preventing from pyruvate 319
over-accumulation. In addition, PEP may be exchanged between the chloroplast and the 320
cytoplasm (exchanged against Pi) through specific transporters (PPT1 and PPT2 in 321
Arabidopsis; for a review, see Linka and Weber 2010). In the chloroplast, PEP is a 322
fundamental precursor of aromatic amino acids and fatty acids (via pyruvate) and activates 323
ADP-glucose synthesis (Ghosh and Preiss 1966). Pyruvate (with glyceraldehyde-3-phosphate) 324
is also the precursor of methylerythritol, a key intermediate of chloroplastic isoprenoid 325
synthesis (Rohmer et al. 1993, 1996). Isoprenoid production is believed to be substantial in 326
illuminated leaves at moderate to high temperature, with the accumulation of 2-C-methyl-D-327
erythritol 2,4-cyclodiphosphate, which may in turn sequestrate most of stromal phosphate 328
(Rivasseau et al. 2009). Therefore, sustaining the chloroplast with PEP molecules of cytosolic 329
origin might be beneficial to maintain stromal pyruvate and Pi contents. 330
The PPDK consumes Pi and synthesises pyrophosphate (PPi), thereby affecting the 331
phospho-balance of the cell. Typically, the cytoplasmic PPDK-catalysed reaction may 332
contribute to maintaining a low cytoplasmic Pi content, for which homeostatic regulation 333
appears critical (Rebeillé et al. 1983, Pratt et al. 2009) and/or pyrophosphate synthesis as an 334
energy donor (Weiner et al. 1987, Dancer et al. 1990) at the expense of ATP. 335
In other words, conversion of pyruvate back to PEP likely play multiple roles upon 336
illumination to: (i) promote PEP resynthesis for PEPc-catalysed oxaloacetate synthesis 337
associated with nitrogen assimilation or chloroplastic isoprenoid metabolism, (ii) impede 338
possible increase in cytoplasmic ATP/Pi ratio (e.g. under large photorespiratory conditions, 339
that generates substantial reducing equivalents in the mitochondrion). In fact, the PEP content 340
appeared tightly correlated to ATP or photosynthetic phosphorylated metabolites and 341
furthermore, the apparent mass action ratio of pyruvate kinase seemed to increase with 342
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17
fructose-1,6-bisphosphate content (Figure 5). PEP resynthesis from pyruvate might thus be 343
involved in catabolic vs photosynthetic adjustments. We nevertheless recognize that the 344
control exerted by pyruvate-to-PEP conversion on the whole respiratory pathway is probably 345
minor since the associated flux is rather small under our experimental conditions. Further 346
work is needed (e.g., characterizing PPDK-mutants) to identify which one of the metabolic 347
roles mentioned above is the most significant. 348
349
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18
Material and methods 350
Plant material and gas-exchange 351
Growth conditions of Xanthium strumarium plants and gas-exchange techniques were already 352
described in Tcherkez et al. (2009). The third or fourth leaf (from the apical bud) was used for 353
all measurements. Detached leaves were placed in a large gas-exchange cuvette connected to 354
the open system Li-Cor 6400 (Lincoln, USA). In all experiments, photosynthesis was allowed 355
to stabilise before labeling and then leaves were labeled for 3 hours (13CO2 and/or 13C-356
pyruvate). 13CO2 (99% 13C) and 12CO2 were purchased at Eurisotop (Saint-Aubin, France) and 357
Air Liquide (Grigny, France), respectively. For NMR analyses, the leaf was instantly frozen 358
at the end of the labeling period with a freeze-spray system: liquid nitrogen was sprayed 359
instantly, that is, 0.1 second after the puncture of the upper wall of the chamber. The leaf 360
sample was then kept at –80°C. 361
362
NMR analyses 363
NMR measurements were carried out as described in Tcherkez et al. (2005) from perchloric 364
acid extracts prepared from ca. 7 g of frozen leaf material. Spectra were obtained using a 365
Bruker spectrometer (AMX 400, Bruker, Wissembourg, France) equipped with a 10-mm 366
multinuclear probe tuned at 100.6 (13C) and 161.9 MHz (31P). The assignment of 13C and 31P 367
resonance peaks was carried out according to Gout et al. (1993) and Bligny et al. (1990), 368
respectively. Peak intensities were normalized to a known amount of the internal reference 369
compound (maleate for 13C and methyl-phosphonate for 31P) that was added to the sample 370
(internal standard). Two types of control were done for NMR analyses: experiments with 371
water (no labeling substrates) and those with 12C-substrates. The latter control was required to 372
normalize the 13C-signal after 13C-labeling so as to calculate the 13C-percentage (% 13C). 373
374
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Liquid-chromatography time-of-flight mass spectrometry (LC-TOF-MS) 375
A 500 µL aliquot of the perchloric extract (see above, NMR analyses) was preleved and stored 376
at –80°C for further use. 10 µL phosphoric acid (50%) were added to the extract and then a 377
double solid phase extraction was carried out, using Strata-X-C and Strata-A-W columns 378
(Phenomenex, Le Pecq, France). Eluates were speed-vac dried and resuspended in water. 379
Samples were injected in the UPLC Acquity equipped with the column UPLC-HSS T3 380
(2.1×100mm, 1.8µm) (Waters, Guyancourt, France) under an ammonium acetate/methanol 381
gradient (99/1-0/100%, 6 min). Electrospray mass spectrometry was carried out with the 382
MicrOTOF II (Bruker Daltonics, Wissembourg, France) with N2 (spray gas) upon negative 383
ionisation. All compounds analysed here were measured against a calibration curve with pure 384
standards purchased at Sigma-Aldrich (Saint-Quentin Fallavier, France). 385
386
Pyruvate Pi dikinase activity 387
The procedure used to determine PPDK leaf activity was inspired from Aoyagi and Bassham 388
(1983) and Nakamoto and Edwards (1983). About 1 g of leaf frozen material was ground at 389
4°C using a cold mortar and 1 mL extracting buffer (0.1 M Tris-HCl, pH 7.5, 10 mM MgCl2, 390
5 mM sodium pyruvate, 2mM K2HPO4, 1 mM EDTA, 1% w/v sodium ascorbate, 10 mM β-391
mercaptoethanol, 1% w/v PVP). The homogenate was filtered through one layer of nylon 392
mesh (30 µm pore size), then briefly centrifugated (1 min at 990 rpm) and desalted with a 393
NAP-5 column (GE Healthcare, Aulnay-sous-bois, France) previously equilibrated with 0.1 394
M Tris-HCl, 10 mM MgCl2, 1 mM EDTA, 1% w/v sodium ascorbate, 10 mM β-395
mercaptoethanol at pH 7.5. PPDK activity was assayed spectrophotometrically (PEPc-MDH 396
coupled assay) as described previously (Edwards et al. 1980). 397
398
Chemicals 399
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Chemicals for assays and isotopic products, 13C-3-pyruvate (99% 13C in C-3) and d-13C-3-400
pyruvate (50% D and 99% 13C in C-3), were purchased at Isotec Sigma-Aldrich. d-12C-401
pyruvate (50% D) (used as an isotopic control for labeling experiments) was synthesised from 402
natural pyruvate incubated for 48 h in deuterium oxide (D2O) at 21°C, after Long and George 403
(1960). In all cases, the final pyruvate concentration used in the experiments was 0.015 mol L-404
1. The solutions were fed to leaves through the transpiration stream. d-13C-3-pyruvate 405
solutions used in experiments were prepared just before the onset of labeling, to minimize the 406
spontaneous keto-enol equilibrium of pyruvate. 407
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21
Acknowledgements
G.T., A.M. and E.B.-F. acknowledge the financial support by the Agence Nationale de la
Recherche, with the project Jeunes Chercheurs, under contract no. JC08-330055.
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References
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Scheible WR, Krapp A, Stitt M (2000) Reciprocal diurnal changes of PEPc expression, cytosolic pyruvate kinase, citrate synthase and NADP-isocitrate dehydrogenase expression regulate organic acid metabolism during nitrate assimilation in tobacco leaves. Plant Cell Environ 23: 1155-1167. Stitt M, Lilley RM, Heldt HW (1982) Adenine nucleotide levels in the cytosol, chloroplasts and mitochondria of wheat leaf protoplasts. Plant Physiol 70: 971-977. Sugiyama T (1973) Purification, molecular and catalytic properties of pyruvate phosphate dikinase from the maize leaf. Biochemistry 12: 2862-2868. Taylor L, Nunes-Nesi A, Parsley K, Leiss A, Leach G, Coates S, Wingler A, Fernie AR, Hibberd JM (2010) Cytosolic pyruvate orthophosphate dikinase functions in nitrogen remobilization during leaf senescence and limits individual seed growth and nitrogen content. Plant J 62: 641-652. Tcherkez G, Cornic G, Bligny R, Gout E, Ghashghaie J (2005) In vivo respiratory metabolism of illuminated leaves. Plant Physiol 138: 1596-1606. Tcherkez G, Cornic G, Bligny R, Gout E, Mahé A, Hodges M (2008) Respiratory metabolism of illuminated leaves depends on CO2 and O2 conditions. Proc Nat Acad Sci USA 105(2): 797-802. Tcherkez G, Mahé A, Gauthier P, Mauve C, Gout E, Bligny R, Cornic G, Hodges M (2009) In folio respiratory fluxomics revealed by 13C isotopic labeling and H/D isotope effects highlight the non-cyclic nature of the tricarboxylic acid "cycle'' in illuminated leaves. Plant Physiol 151: 620-630. Tovar-Mendez A, Miernyk JA, Randall DD (2003) Regulation of pyruvate dehydrogenase complex activity in plant cells. Eur J Biochem 270: 1043-1049. Weiner H, Stitt M, Heldt HW (1987) Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves. Biochim Biophys Acta - Bioenergetics 893: 13-21.
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Figure legends Figure 1. Simplified metabolic scheme showing the potential fates of pyruvate and the labeling pattern after 13C-3-pyruvate addition to illuminated leaves. The labeled position in C-3-pyruvate and molecules downstream through pyruvate dehydrogenase (PDH, 1), alanine aminotransferase (AlaAT, 2) and the tricaboxylic acid cycle is shown in black. Labeled C-atom positions coming from pyruvate Pi dikinase (PPDK, 4) or reverse pyruvate kinase (PK, 3) activity are shown in grey. The small grey labeled C-atom accounts for the fumarate/malate equilibrium that interconverts C-2 and C-3 atoms due to molecular symmetry. This scheme does not take into account multiple turns of the tricarboxylic acid cycle and assumes little conversion of 2-oxoglutarate to fumarate (dashed arrow, see main text and Tcherkez et al. 2009). AspAT, aspartate aminotransferase; Fase, fumarase; MDH, malate dehydrogenase; PEPc, phosphoenolpyruvate carboxylase. Figure 2. Labeling patterns in visible C-atom positions in metabolites, detected with 13C-NMR after 13C-3-pyruvate or/and 13CO2 labeling of illuminated X. strumarium leaves for 3 hours, under 400 µmol mol-1 CO2, 21% O2, 23.5°C and 300 µmol m-2 s-1 PAR. In each cell, the colour represents the positional 13C-abundance in % 13C (colour scale on the right). C-atom positions are numbered according to the international chemical nomenclature (for glutamate, see Figure 3). For glucose, a and b stand for α and β isomers. For the C-3 atom position in oxalo acetate, the two isomeric forms are distinguished: C=O (keto) and C–OH (enol). The average abundance chosen here (1.1%) corresponds to the natural 13C-abundance. 13C-abundance values shown here are means of three replicates. The colour scale is here restricted to 10% so as to make low 13C-enrichments visible. Fumarate nevers appear labeled due to isotopic dilution caused by its very large content in X. strumarium leaves (near 20 mmol m-2, not shown here). The C-5 atom position in glutamate and C-4 atom position in malate are not shown here because of insufficient signal intensity or resolution. Cit, citrate; Fru, fructose (pyranic form); Fru-f, fructose (furanic form); Fum, fumarate; GABA, γ-aminobutyrate; Glc 1b, 1a, C-1 position of α and β enantiomers of glucose; Icit, isocitrate; Mal, malate; 2-OG, 2-oxoglutarate; OAA, oxalo acetate; Pyr, pyruvate; Suc-G, glucosyl moiety of sucrose; Suc-F, fructose moiety of sucrose; Succ, succinate. Figure 3. Positional 13C-NMR signals in glutamate, after 13C-3-pyruvate or/and 13CO2 labeling of illuminated X. strumarium leaves for 3 hours, under 400 µmol mol-1 CO2, 21% O2, 23.5°C and 300 µmol m-2 s-1 PAR. A, C-atom numbering in glutamate. B, Positional 13C-abundance (in % 13C) in glutamate (redrawn from Figure 2). The natural 13C-abundance (1.1%) is indicated with the horizontal dashed line. The major origin of 13C-labeling in each position is indicated with arrows, as discussed in the main text. C, {13C-13C} spin-spin interactions in glutamate. For a given labeled position, numbers between arrows indicate the percentage of having 13C-labeling in the neighbour carbon atoms. Bidirectional arrows indicate that two {13C-13C} interactions were obtained, demonstrating there were two interacting, labeled neighbour carbons. For example, in C-3 glutamate after pyruvate labeling, the percentage of 13C in C-3 that was accompanied by 13C in C-2 and C-4 was 62%. In 2-oxoglutarate, {13C-13C} spin-spin interactions were only visible (distinguishable from the background) in C-4 because of the larger 13C-enrichment in that position. The value of 1% (glutamate C-2 upon 13C-pyruvate labeling) is arbitrary and corresponds to the natural interaction (13C natural abundance of 1.1%). Bidirectional half-arrows indicate that a single {13C-13C} interaction occurred, on one side only of the C-atom position considered. From top to bottom, interactions values indicated here are that observed after 13C-pyruvate labeling, 13CO2 labeling and after double labeling, in that order.
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Figure 4. The 33.5-34.5 ppm region of the NMR-spectrum showing the 13C-signal of the C-4 atom position in glutamate in illuminated X. strumarium leaves after deutero-13C-3-pyruvate (A) or protio-13C-3-pyruvate (B) labeling for 3 hours, under 400 µmol mol-1 12CO2, 21% O2, 23.5°C and 300 µmol m-2 s-1 PAR. {13C–13C} and {13C–D} single interactions are shown with dashed (JC–C) and dash-dotted lines (JC–D), respectively. In A, the deuterium isotope effect on the chemical shift is indicated with the arrow labeled 'αD' (single deuteration, CHD) and 'αD
2' (double deuteration, CD2). Peaks associated with the double deuteration are labeled with dotted vertical lines (other peaks beyond 33.5 ppm are not shown here). The area integration indicates that the proportion of deuterated 13C atoms is 33% and the 13C-interacting 13C-atoms is 5.0% of 13C-atoms. In B, the interaction with neighour 13C-atoms represents 4.5%. Figure 5. Leaf metabolic amounts (A, B) and pyruvate Pi dikinase (PPDK) activity (C) in X. strumarium, in the dark and in the light (400 µmol mol-1 CO2, 21% O2, 23.5°C, 300 µmol mol-1 PAR, no labeling). Phosphoenolpyruvate (PEP) content was determined by quantitative 31P-NMR from perchloric extracts. ATP/ADP and ATP/AMP ratios were measured on the same extracts with liquid-chromatography/time-of-flight mass spectrometry. In vitro PPDK activity (nmol PEP m-2 s-1) was measured on desalted leaf extracts using a phosphoenolpyruvate carboxylase-malate dehydrogenase coupled enzymatic assay. Values are mean±SE (n=3). Figure 6. Natural variations in phosphometabolite content in illuminated X. strumarium leaves, under 400 µmol mol-1 CO2, 21% O2, 23.5°C, 300 µmol mol-1 PAR, fed with pyruvate through the transpiration stream. A, array representation of the phosphometabolome. Metabolites were quantified by liquid chromatography/time-of-flight mass spectrometry (ATP, ADP, AMP, UDP-Glc, ADP-Glc, UTP, UDP) and 31P-NMR (others). The hierachical clustering (left) was carried out with the cosine correlation on mean-centered metabolite contents. The range of the natural variation of phosphometabolites is ±50% (color scale on top). Column numbers on top (1-6) are biological replicates. DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; GPC, glyceryl-phosphophorylcholine; P-Cho, phosphoryl-choline; PEA, phosphatidyl-ethanolamine; P-EA, 3-phosphoryl-ethanolamine; PGA, phosphoglycerate; RuBP, ribulose-1,5-bisphosphate; Pi, inorganic phosphate. B, mass action ratio of phosphometabolites on the pyruvate kinase (PEP.ADP/ATP, open symbols) and pyruvate Pi dikinase (PEP.AMP/ATP, closed symbols) as a function of fructose-1,6-bisphosphate. C, PEP content relative to fructose-1,6-bisphosphate (closed symbols) or AMP (open symbols) as a function of UDP-Glc/UTP ratio, an indicator of sucrose synthesis. In both B and C, metabolite concentrations are given in µmol m-2. Continuous and dashed lines represent the apparent trends of the curves.
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Figure 1. Simplified metabolic scheme showing the potential fates of pyruvate and the
labeling pattern after 13C-3-pyruvate addition to illuminated leaves. The labeled position
in C-3-pyruvate and molecules downstream through pyruvate dehydrogenase (PDH, 1),
alanine aminotransferase (AlaAT, 2) and the tricaboxylic acid cycle is shown in black.
Labeled C-atom positions coming from pyruvate Pi dikinase (PPDK, 4) or reverse
pyruvate kinase (PK, 3) activity are shown in grey. The small grey labeled C-atom
accounts for the fumarate/malate equilibrium that interconverts C-2 and C-3 atoms due to
molecular symmetry. This scheme does not take into account multiple turns of the
tricarboxylic acid cycle and assumes little conversion of 2-oxoglutarate to fumarate
(dashed arrow, see main text and Tcherkez et al. 2009). AspAT, aspartate
aminotransferase; Fase, fumarase; MDH, malate dehydrogenase; PEPc,
phosphoenolpyruvate carboxylase.
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Figure 2. Labeling patterns in visible C-atom positions in metabolites, detected with 13C-NMR after 13C-3-pyruvate
or/and 13CO2 labeling of illuminated X. strumarium leaves for 3 hours, under 400 µmol mol-1 CO2, 21% O2, 23.5°C and
300 µmol m-2 s-1 PAR. In each cell, the colour represents the positional 13C-abundance in % 13C (colour scale on the right).
C-atom positions are numbered according to the international chemical nomenclature (for glutamate, see Figure 3). For
glucose, a and b stand for a and b isomers. For the C-3 atom position in oxalo acetate, the two isomeric forms are
distinguished: C=O (keto) and C–OH (enol). The average abundance chosen here (1.1%) corresponds to the natural 13C-
abundance. 13C-abundance values shown here are means of three replicates. The colour scale is here restricted to 10% so
as to make low 13C-enrichments visible. Fumarate nevers appear labeled due to isotopic dilution caused by its very large
content in X. strumarium leaves (near 20 mmol m-2, not shown here). The C-5 atom position in glutamate and C-4 atom
position in malate are not shown here because of insufficient signal intensity or resolution. Cit, citrate; Fru, fructose
(pyranic form); Fru-f, fructose (furanic form); Fum, fumarate; GABA, g-aminobutyrate; Glc 1b, 1a, C-1 position of a and
b enantiomers of glucose; Icit, isocitrate; Mal, malate; 2-OG, 2-oxoglutarate; OAA, oxalo acetate; Pyr, pyruvate; Suc-G,
glucosyl moiety of sucrose; Suc-F, fructose moiety of sucrose; Succ, succinate.
0
1.1
10
13C-Pyr
13CO2
Both
Other amino acids Organic acids amino acids Sugars TCA-derived
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C-1 C-2 C-3 C-4
Pos
ition
al 13
C a
bund
ance
(%
)
0
5
10
15
20
C-atom position in glutamate
C-1 C-2 C-3 C-4 C-1 C-2 C-3 C-4
13C-Pyr 13CO2Both
TCA
PPDK-PEPcH13CO3
− PEPc
13C-PEPPEPc
A
B
C
Figure 3. Positional13C-NMR signals in glutamate, after13C-3-pyruvateor/and13CO2 labeling of illuminatedX. strumarium leaves for 3 hours, under400 µmol mol-1 CO2, 21% O2, 23.5°C and 300 µmol m-2 s-1 PAR. A, C-atom numbering in glutamate. B, Positional 13C-abundance (in % 13C) in glutamate (redrawn fromFigure 2). The natural13C-abundance (1.1%) is indicated with the horizontal dashed line. The major origin of 13C-labeling ineach position is indicated with arrows, as discussed in the main text. C, { 13C-13C} spin-spin interactions in glutamate. For a given labeled position,numbers between arrows indicate the percentage of having13C-labeling inthe neighbour carbon atoms. Bidirectional arrows indicate that two {13C-13C} interactions were obtained, demonstrating there were two interacting,labeled neighbour carbons. For example, in C-3 glutamate after pyruvate labeling, the percentage of 13C in C-3 that was accompanied by 13C in C-2and C-4 was 62%. In 2-oxoglutarate, {13C-13C} spin-spin interactions were only visible (distinguishable from the background) in C-4 because of the larger13C-enrichment in that position. The value of 1% (glutamate C-2 upon13C-pyruvate labeling) is arbitrary and corresponds to the natural interaction (13C natural abundance of 1.1%). Bidirectional half-arrows indicate that a single {13C-13C} interaction occurred, on one side only of the C-atomposition considered. From top to bottom, interactions values indicated hereare that observed after13C-pyruvate labeling, 13CO2 labeling and afterdouble labeling, in that order.
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34.5 34.0 33.5
10 Hz
J C-C
αD
JC-DJ C-C
Ad-13C-3-pyruvate
B13C-3-pyruvate
αD2
Figure 4. The 33.5-34.5 ppm region of the NMR-spectrum showing the13C-signal of the C-4 atomposition in glutamate in illuminatedX. strumarium leaves after deutero-13C-3-pyruvate (A) orprotio-13C-3-pyruvate (B) labeling for 3 hours, under 400 µmol mol-1 12CO2, 21% O2, 23.5°C and300 µmol m-2 s-1 PAR. {13C–13C} and {13C–D} single interactions are shown with dashed (JC–C) and dash-dotted lines (JC–D), respectively. In A, the deuterium isotope effect on the chemical shift is indicated with the arrow labeled 'αD' (single deuteration, CHD) and 'αD
2' (double deuteration, CD2).Peaks associated with the double deuteration are labeled with dotted vertical lines (other peaks beyond 33.5 ppm are not shown here). The area integration indicates that the proportion ofdeuterated13C atoms is 33% and the13C-interacting13C-atoms is 5.0% of 13C-atoms. In B, theinteraction with neighhour13C-atoms represents 4.5%.
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Figure 5. Leaf metabolic amounts (A, B) and pyruvate Pi dikinase (PPDK) activity (C) in X. strumarium, in the dark and
in the light (400 µmol mol-1 CO2, 21% O2, 23.5°C, 300 µmol mol-1 PAR, no labeling). Phosphoenolpyruvate (PEP)
content was determined by quantitative 31P-NMR from perchloric extracts. ATP/ADP and ATP/AMP ratios were measured
on the same extracts with liquid-chromatography/time-of-flight mass spectrometry. In vitro PPDK activity (nmol PEP m-2
s-1) was measured on desalted leaf extracts using a phosphoenolpyruvate carboxylase-malate dehydrogenase coupled
enzymatic assay. Values are mean±SE (n=3).
Day Night
Leaf
conte
nt
(µm
ol m
-2)
0
10
20
30
40
50PEP
Pyruvate
Day Night
Leaf
ratio
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
ATP/ADP
ATP/AMP
Day Night
In v
itro
PP
DK
activity (
nm
ol m
-2 s
-1)
0
10
20
30
40
50A B C
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Fru-1,6-bisP (µmol m-2
)
0.0 0.5 1.0 1.5 2.0
Ratio (
µm
ol m
-2)
0.0
0.5
1.0
1.5
2.0PEP.AMP/ATP
PEP.ADP/ATP Col 10 vs Col 11
UDPG/UTP
0 1 2 3 4
Ratio
0
1
2
3
4
5
6
PEP/FBP
PEP/AMP
B C
Figure 6. Natural variations in phosphometabolite content in illuminated X.
strumarium leaves, under 400 µmol mol-1 CO2, 21% O2, 23.5°C, 300 µmol
mol-1 PAR, fed with pyruvate through the transpiration stream. A, array
representation of the phosphometabolome. Metabolites were quantified by
liquid chromatography/time-of-flight mass spectrometry (ATP, ADP, AMP,
UDP-Glc, ADP-Glc, UTP, UDP) and 31P-NMR (others). The hierachical
clustering (left) was carried out with the cosine correlation on mean-centered
metabolite contents. The range of the natural variation of phosphometabolites
is 50% (color scale on top). Column numbers on top (1-6) are biological
replicates. DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-
phosphate; GPC, glyceryl-phosphophorylcholine; P-Cho, phosphoryl-choline;
PEA, phosphatidyl-ethanolamine; P-EA, 3-phosphoryl-ethanolamine; PGA,
phosphoglycerate; RuBP, ribulose-1,5-bisphosphate; Pi, inorganic phosphate.
B, mass action ratio of phosphometabolites on the pyruvate kinase
(PEP.ADP/ATP, open symbols) and pyruvate Pi dikinase (PEP.AMP/ATP,
closed symbols) as a function of fructose-1,6-bisphosphate. C, PEP content
relative to fructose-1,6-bisphosphate (closed symbols) or AMP (open
symbols) as a function of UDP-Glc/UTP ratio, an indicator of sucrose
synthesis. In both B and C, metabolite concentrations are given in µmol m-2.
Continuous and dashed lines represent the apparent trends of the curves.
I
II
III
A -0.5 +0.5
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